Download - Synergistic Activation of HIV1 Expression by Deacetylase Inhibitors and Prostratin: Implications for Treatment of Latent Infection

Synergistic Activation of HIV-1 Expression byDeacetylase Inhibitors and Prostratin: Implications forTreatment of Latent InfectionSophie Reuse1., Miriam Calao1., Kabamba Kabeya2, Allan Guiguen1, Jean-Stephane Gatot1, Vincent

Quivy1, Caroline Vanhulle1, Aurelia Lamine3, Dolores Vaira4, Dominique Demonte1, Valerie Martinelli1,

Emmanuelle Veithen1, Thomas Cherrier5, Veronique Avettand6, Solene Poutrel3, Jacques Piette7, Yvan

de Launoit8, Michel Moutschen4, Arsene Burny1, Christine Rouzioux6, Stephane De Wit2, Georges

Herbein9, Olivier Rohr5, Yves Collette10, Olivier Lambotte3, Nathan Clumeck2, Carine Van Lint1*

1 Laboratory of Molecular Virology, Institut de Biologie et de Medecine Moleculaires (IBMM), Universite Libre de Bruxelles (ULB), Gosselies, Belgium, 2 Service des Maladies

Infectieuses, CHU St-Pierre, Universite Libre de Bruxelles (ULB), Bruxelles, Belgium, 3 Faculte de Medecine Paris-Sud, INSERM U802, Bicetre, France, 4 AIDS Reference

Center, University of Liege (ULg), Liege, Belgium, 5 Virology Institute, INSERM U575, Strasbourg, France, 6 Service de Virologie, EA3620, Universite Paris-Descartes, AP-HP,

Hopital Necker-Enfants-Malades, Paris, France, 7 Laboratory of Virology and Immunology, GIGA-R, University of Liege (ULg), Liege, Belgium, 8 Institut de Biologie de Lille,

Institut Pasteur de Lille, UMR 8117 CNRS, BP447, Universite de Lille 1, Lille, France, 9 Department of Virology, EA3186, IFR133, Franche-Comte University, Hopital Saint-

Jacques, Besancon, France, 10 Centre de Recherche en Cancerologie de Marseille, INSERM UMR 599, Marseille, France

Abstract

The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy(HAART)-treated infected individuals, represents a major hurdle to virus eradication. Activation of HIV-1 gene expression inthese cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool oflatent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we heredemonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs)combined with prostratin, a non-tumor-promoting nuclear factor (NF)- kB inducer. In J-Lat cells, we showed that thissynergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population.A combination of prostratin+HDACI synergistically activated the 59 Long Terminal Repeat (5’LTR) from HIV-1 Major groupsubtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays.Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-kB and degradation ofcytoplasmic NF-kB inhibitor, IkBa . Moreover, the combined treatment prostratin+HDACI caused a more pronouncednucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This morepronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatmentprostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5’LTR and both initiated andelongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8+-depletedperipheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combinedtreatment reactivated viral replication in resting CD4+ T cells isolated from similar patients. Our results suggest thatcombinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1reservoirs in HAART-treated patients.

Citation: Reuse S, Calao M, Kabeya K, Guiguen A, Gatot J-S, et al. (2009) Synergistic Activation of HIV-1 Expression by Deacetylase Inhibitors and Prostratin:Implications for Treatment of Latent Infection. PLoS ONE 4(6): e6093. doi:10.1371/journal.pone.0006093

Editor: Peter Sommer, Institut Pasteur Korea, Republic of Korea

Received October 15, 2008; Accepted May 7, 2009; Published June 30, 2009

Copyright: � 2009 Reuse et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: We acknowledge grant support from the Belgian Fund for Scientific Research (FRS-FNRS, Belgium), the Televie-Program of the FRS-FNRS, the Action deRecherche Concertee du Ministere de la Communaute Francaise (Universite Libre de Bruxelles, ARC program no. 04/09-309), the Programme d’Excellence ‘‘Cibles’’of the Region Wallonne, the Region Wallonne (Program WALEO 021/5110), the Internationale Brachet Stiftung, the Interreg III program (Intergenes project), theAgence Nationale de Recherches sur le SIDA (ANRS), the Sidaction and the Fondation pour la Recherche Medicale (FRM, France). S.R. is fellow of the Belgian Fondspour la Recherche dans l’Industrie et l’Agriculture (FRIA). M.C. is fellow of the Belgian Fonds pour la Recherche dans l’Industrie et l’Agriculture (FRIA) and of theTelevie-Program of the FRS-FNRS. A.G. is supported by a post-doctoral fellowship from the Region Wallonne (Program WALEO2616295). J.-S.G. is supported by apostdoctoral fellowship from the ULB (Universite Libre de Bruxelles, ARC program no. 04/09-309). C.V.L. is Directeur de Recherches of the FRS- FNRS. The fundershad no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

. These authors contributed equally to this work.

Introduction

HIV-1 infection can be treated effectively in many patients in

the developed world, using combinations of antiretroviral

therapeutics, called Highly Active Anti-Retroviral Therapy

(HAART). However, despite prolonged treatment with HAART,

the persistence of HIV-1 reservoirs harboring transcriptionally

silent but replication-competent proviruses represents the major

PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e6093

hurdle to virus eradication. These latently-infected cells are a

permanent source for virus reactivation and lead to a rebound of

the viral load after interruption of HAART. Therefore, current

anti-HIV-1 research efforts are increasingly focused on strategies

aimed at reducing the size of these persistent reservoirs of latent

HIV-1 by forcing viral gene expression. This kind of strategy

would allow latently-infected cells to die from viral cytopathic

effects or host cytolytic effector mechanisms following viral

reactivation, while the antiretroviral therapy would prevent

spreading of the infection by the neosynthetized virus [1,2].

Acetylation level of histone and non-histone proteins, controlled

by deacetylases (HDACs) and acetyltransferases (HATs), is a key

element regulating HIV-1 transcription. In agreement, we have

previously reported that treatment of latently HIV-1-infected cell

lines with HDAC inhibitors (HDACIs) induces viral transcription

and remodeling of the repressive nucleosome nuc-1, located

immediately after the HIV-1 transcription start site under latency

conditions [3,4]. Similar results were observed in transiently or

stably transfected HIV-1 Long Terminal Repeat (LTR) reporter

constructs [5,6,7], and on in vitro chromatin-reconstituted HIV-1

templates [8,9]. Based on these observations, administration of

HDACIs together with efficient HAART has been proposed as an

inductive adjuvant therapy for the decay of latent reservoirs

[10,11,12,13]. The Margolis group has reported that VPA

(valproic acid), in the presence of IL-2, induces rescue of

replication-competent HIV-1 from purified resting CD4+ T cells

obtained from HAART-treated patients with undetectable viral

load [14]. Later, in a clinical trial performed by same group, four

patients receiving HAART and the viral entry inhibitor enfuvirtide

were given VPA for three months, and a modest but significant

decrease in the frequency of latently-infected cells was noted in

three of the four patients [15]. However, given that at least two

studies have demonstrated that intensification of anti-HIV therapy

decreases the half-life of this population [16,17], it is unclear

whether VPA or intensification of HAART with enfuvirtide was

the critical factor for the decay of the latent reservoir. Recent

reports have failed to show a decay of resting CD4+ T cell

infection in patients who were prescribed VPA for clinical reasons

while receiving standard HAART [18,19,20]. These results led to

question the therapeutic potential of VPA, at least when used

alone, to reduce the size of the latent HIV-1 reservoirs.

We have previously demonstrated a strong synergistic activation

of HIV-1 promoter activity by the HDACI trichostatin A (TSA)

and the NF-kB inducer TNFa in the postintegration latency

model cell line U1 [3,21], suggesting that combinations of two

independent factors (NF-kB and chromatin) involved in HIV-1

reactivation from latency might be potent tools to decrease the

pool of latently-infected reservoirs. However, because of their

toxicity, the therapeutic use of TNFa and TSA is not possible.

Here, we investigated the HIV-1 reactivating potential of a

treatment combining HDACIs used in human clinical trials or

therapies (such as VPA and suberoylanilide hydroxamic acid

[SAHA]) and an NF-kB inducer with no tumor-promoting effects,

prostratin. This compound is an inducer of protein kinase C

activity which stimulates HIV-1 expression in latently-infected

lymphoid and myeloid cell lines and primary cells

[22,23,24,25,26,27,28,29] with minimal effects on the immune

system [26] and causing minimal cell cycle progression [26,29].

Moreover, prostratin also inhibits de novo HIV-1 infection via

posttranscriptional downregulation of the cellular HIV-1 receptors

CD4 and CXCR4 (CXC chemokine receptor 4) [24,27,30,31,32].

Therefore, the antimitogenic property of prostratin coupled with

its dual activity on HIV-1 infection (inhibition of viral infection

and upregulation of latent provirus expression), its relatively non-

toxic behavior and its potential widespread effect on different

HIV-1 reservoirs, make this compound a good candidate for viral

purging. Although the suitability of prostratin for use in humans is

unknown since preclinical testing in compliance with Food and

Drug Administration regulations is reportedly still underway

[12,33], preliminary pharmacokinetic studies are encouraging as

they show that 5 of 5 mice survived with no obvious effects at

100 mmol/Kg intragastric dose of prostratin with plasma concen-

trations reaching up to 1.42 mmol/L [24]. These results are not

surprising as plant extracts of Homalanthus nutans (a source of

prostratin) have already been used by the Samoan healers to treat

individuals with certain medical conditions such as hepatitis

[34,35].

Here, we demonstrated that a combination of prostratin+VPA

or prostratin+SAHA reactivated more efficiently than each

compound alone HIV-1 production both in several latently-

infected cell lines (U1 and J-Lat clones) and in CD8+-depleted

peripheral blood mononuclear cells (PBMCs) isolated from HIV-

1-infected patients receiving HAART and with undetectable viral

load. Mechanistically, HDACI increased prostratin-induced NF-

kB activation and potentiated nuc-1 remodeling. Moreover,

prostratin+HDACI combined treatment caused a synergistic

activation of HIV-1 transcriptional initiation and elongation.

Our results constitute a proof-of-concept for the coadministration

of two different types of therapeutically promising HIV-1 inducers

(one acting on the NF-kB pathway and the other acting on the

protein acetylation status) together with HAART as a therapeutic

perspective to decrease the pool of latent HIV-1 reservoirs.

Results

Synergistic activation of HIV-1 production by prostratinand clinically used HDACIs

HDACIs present several advantages for HIV-1 purging

strategies. They do not induce proliferation or activation of T

cells [36,37], act in a broad spectrum of cell lines and potently

repress CXCR4 chemokine receptor expression and function [38].

Moreover, whereas TSA has a high toxicity and a costly and

poorly efficient production [39], several HDACIs are safely

administered for other diseases [40,41,42,43,44,45] or tested in

clinical trials as anticancer drugs [46]. HDACIs are specific for

class I and II or class III HDACs and have been subdivided into

four distinct groups based on their structural characteristics:

hydroxamic acids; short-chain fatty acids; synthetic benzamide

derivatives; cyclic tetrapeptides/epoxides [39,46,47].

For various HDACIs, we determined an optimal concentration

in terms of both their cellular toxicity and their HIV-1 reactivation

potential by measuring cell viability (Figure S1 and Text S1) and

induction of p24 production in HDACI-treated U1 cells (Figure 1).

The latently HIV-1-infected monocytic cell line U1 is one of the

most studied cellular models of postintegration latency. All the

inhibitors of class I and II HDACs we tested increased core

antigen levels (p24) in the U1 cellular supernatants, independently

of the structural group the HDACIs belong to (Figure 1A, 1B, 1C

and 1D). In the short-chain fatty acid group, VPA was a weaker

inducer of HIV-1 p24 production than NaBut (sodium butyrate)

(Figure 1A). MS-275 induced viral p24 antigen production 112-

fold at a concentration of 20 mM (Figure 1B). Among the cyclic

tetrapeptide group, Apicidin at 1 mM exhibited the highest

reactivation of viral production (Figure 1C). All HDACIs from

the hydroxamic acid group were more efficient than TSA to

induce HIV-1 p24 antigen production (Figure 1D). Splitomycin

and Sirtinol, which are specific inhibitors of class III HDACs, had

no effect on p24 production (Figure 1A and 1B, respectively).

Reactivation of Latent HIV-1


Among the HDACIs tested, VPA, NaBut and SAHA are

already in clinical use to reduce epileptic seizures [43], to treat

sickle cell anemia and beta-thalassemia [41] and for the treatment

of cutaneous T-cell lymphoma [42,48,49], respectively, whereas

others, such as MS-275 [50], are in clinical trials. We tested

whether these HDACIs, already used in human therapy and thus

promising for AIDS treatment, synergistically reactivated HIV-1

p24 antigen production in U1 cells when combined with TNFa,

the tumor-promoting phorbol ester PMA (phorbol myristate

acetate) or the non-tumor-promoting phorbol ester prostratin

(Figure 2A, 2B and 2C). Two activators synergize when their

combination produces a level of activation that is greater than the

sum of the effects produced with the individual activators. HDACI

concentrations were chosen according to our reactivation results

(Figure 1) and our cytotoxicity results (Figure S1 and Text S1).

HIV-1 p24 antigen production was synergistically activated by

each TNFa+HDACI cotreatment (Figure 2A). Similarly, we

observed important synergistic activations of virus production

with each combination of the phorbol esters PMA (Figure 2B) or

prostratin (Figure 2C) with an HDACI, except for MS-275.

In order to extend the results obtained in the U1 monocytic

latency model cell line, we decided to confirm our results in the

Jurkat CD4+ T-cell-based model of HIV-1 postintegration latency

termed J-Lat [51]. J-Lats are T-cell clones containing a single, full-

length integrated HIV-1 provirus with the green fluorescent protein

(GFP) open reading frame in place of the nef gene, thus permitting

epifluorescence monitoring of viral transcriptional activity. Under

basal conditions, little or no GFP expression is detected and

transcriptional activation of the latent provirus leads to GFP

expression, which can be detected at the single-cell level by flow

cytometry. In contrast to several other HIV-1 postintegration model

cell lines (including the U1 cell line [52]), the J-Lat clones do not

contain mutations in the HIV-1 tat (trans-activator of transcription)

gene or TAR element (trans-activation response element). We

observed important synergistic activations of viral production in the

two J-Lat clones tested, the J-Lat 15.4 (Figure 2D, 2E and 2F) and

the J-Lat 8.4 (Figure 2G, 2H and 2I) cells, with each combination of

TNFa (Figure 2D and 2G), PMA (Figure 2E and 2H) or prostratin

(Figure 2F and 2I) with an HDACI. Of note, both PMA and

prostratin synergistically activated virus production with MS-275 in

the J-Lat clones 15.4 and 8.4 in contrast to what we observed in the

U1 cell line, indicating that the combinations can present a certain

specificity and underlining the importance of testing the different

combinations.

Our results showed that the non-tumor-promoting phorbol ester

prostratin synergistically reactivated HIV-1 production with

clinically used HDACIs (VPA, SAHA and NaBut) in monocytic

and lymphocytic postintegration latency model systems, the two

major cellular reservoirs in the natural host. Importantly, we

demonstrated using human uninfected CD8+-depleted PBMCs

that prostratin did not increase the cytotoxicity of these HDACIs,

except for the MS-275 which exhibited a slight increased cellular

toxicity when combined with prostratin (Figure S2, compare panel

A and B; Text S2).

Cotreatment with prostratin+HDACI induces HIV-1expression in a higher proportion of J-Lat T cells than thedrugs alone

In order to assess if the synergistic effect observed with a

prostratin+HDACI cotreatment on viral p24 antigen production

was due to an enhanced HIV-1 expression from those cells whose

transcription was already reactivated by the drugs used alone or to

the recruitment of unresponsive cells into the responding

population, we used J-Lat T cells. As these cells harbor a full-

Figure 1. Dose-dependent effects of various HDACIs on HIV-1production. U1 cells were mock-treated or treated with HDACIs: short-chain fatty acids (A), benzamides (B), cyclic tetrapeptides (C),hydroxamic acids (D). At 24 h posttreatment, viral production wasestimated by measuring CA-p24 antigen concentration in culturesupernatants. The mock-treated value was arbitrarily set at a value of 1.Each point is the mean from three separate experiments performed intriplicate. SE are intentionally not represented on the graph for clarityreasons.doi:10.1371/journal.pone.0006093.g001



Figure 2. Synergistic activation of HIV-1 production by prostratin and clinically used HDACIs. U1 (panels A, B and C), J-Lat 15.4 (panelsD, E and F) or J-Lat 8.4 (panels G, H and I) cells were mock-treated or treated with TNFa (10 ng/ml) (panels A, D and G), PMA (20 nM) (panels B, Eand H), prostratin (5 mM) (panels C, F and I) alone or in combination with different HDACIs [VPA (2.5 mM), SAHA (2.5 mM), TSA (500 nM), NaBut(5 mM) or MS-275 (5 mM)]. At 24 h posttreatment, viral production was estimated by measuring CA-p24 antigen concentration in culturesupernatants. The mock-treated value was arbitrarily set at a value of 1. Each value is the mean6SE from two (for the J-Lat 15.4 and 8.4 cell lines) orthree (for the U1 cell lines) separate experiments performed in triplicate.doi:10.1371/journal.pone.0006093.g002



length latent HIV-1 provirus containing GFP in place of nef,

transcriptional activation of the latent provirus can be readily

detected in individual cells by flow cytometry. As shown in

Figure 3, in the absence of stimulation, the J-Lat 15.4 and 8.4 cells

expressed no GFP, indicating a blockage of viral transcription.

Stimulation of the J-Lat 15.4 cells with prostratin, VPA, SAHA,

TSA, NaBut and MS-275 induced GFP expression in a small

proportion of cells (0.20%, 0.03%, 0.09%, 1.90%, 1.80% and

0.02% of cells, respectively) (Figure 3A). Importantly, the

proportion of J-Lat 15.4 cells displaying GFP epifluorescence

was strongly and synergistically increased by the prostratin+H-

DACI cotreatments (Figure 3A). Similar results were obtained

with the J-Lat clone 8.4 (Figure 3B and Figure S3).

These results indicated that, in the Jurkat CD4+ T-cell-based J-

Lat clones, the prostratin+HDACI combinatorial treatment

caused the synergistic recruitment of unresponsive J-Lat cells into

the expressing cell population.

Synergistic activation by prostratin and VPA of LTRactivity from most of HIV-1 subtypes of the HIV-1 major(M) group

The HIV-1 group M isolates, which are responsible for more

than 99% of all infections, have diversified during their worldwide

spread. These isolates have been grouped according to their

genomic sequences and are divided into 9 distinct subtypes termed

A, B, C, D, F, G, H, J and K that can be further divided into sub-

subtypes [53]. Moreover, when an identical recombinant virus is

identified in at least three epidemiologically unlinked people and is

characterized by full-length genome sequencing, it can be

designated as a circulating recombinant form (CRF) [54]. More

than 20 CRFs have been reported such as CRF01_AE (originally

defined as subtype E) or CRF02_AG, recombinations of subtype A

and E or A and G, respectively [53,54].

Functional distinctions in LTR architecture among HIV-1

subtypes have been identified, thus raising the possibility that

regulatory divergence among the subtypes of HIV-1 has occurred.

To examine the impact of these differences among the LTR

sequences on the prostratin+VPA synergism, we performed transient

transfections of Jurkat cells with reporter luciferase constructs

containing LTRs from subtypes A, B, C1, D, F, G, and from

CRF01_AE and CRF02_AG. Transfected LTRs were assayed for

their responsiveness to prostratin alone, to VPA alone, or to both

agents in combination. Results presented in Figure 4 show the

induction for each subtype (obtained by dividing the induced

luciferase activities of subtype X by the basal activity of this same

subtype X) in order to eliminate subtype-specific differences in basal

activity of the LTRs. LTR activity of each subtype tested was induced

by prostratin alone from 5.4- to 17-fold and by VPA alone from 2.8-

to 7.9-fold depending on the subtype (Figure 4). Importantly,

prostratin and VPA together synergized (inductions from 14- to 72-

fold) to activate all the subtype LTRs, except the subtype G LTR.

In conclusion, we showed that the combination prostratin+VPA

synergistically activated transcription from LTRs belonging to

several group M subtypes, including subtypes A, B, C, which

represent the most prevalent HIV-1 genetic forms (subtype C

accounting for almost 50% of all HIV-1 infections worldwide)

[54]. These results were reminiscent of our previous studies using

TNFa and TSA [3].

VPA potentiates prostratin-induced nuclear NF-kB DNA-binding activity and cytoplasmic IkBa degradation

In order to assess the molecular mechanisms responsible for the

synergistic reactivation of HIV-1 production we observed

following a prostratin+VPA cotreatment, we analyzed the effects

of VPA on prostratin-induced NF-kB DNA-binding activity. To

this end, we performed electrophoretic mobility shift assays

(EMSAs) using an HIV-1 NF-kB probe and nuclear extracts from

Jurkat T cells (Figure 5A). As expected, a rapid and strong NF-kB

Figure 3. Prostratin+HDACI cotreatment induces HIV-1 expres-sion in a higher proportion of cells than the drugs alone. J-Lat15.4 (panel A) or 8.4 (panel B) cells were mock-treated or treated withprostratin (5 mM), alone or in combination with different HDACIs [VPA(2.5 mM), SAHA (2.5 mM), TSA (500 nM), NaBut (5 mM) or MS-275(5 mM)]. At 24 h posttreatment, cells were analyzed by FACS for GFPexpression and results (percentage of GFP-positive cells) are represent-ed as histograms. Each value is the mean6SE from two separateexperiments performed in duplicate.doi:10.1371/journal.pone.0006093.g003



DNA-binding activity was observed in response to a 30 min

treatment with TNFa (Figure 5A, lane 2) and VPA alone caused

no induction of NF-kB binding activity even after a 90 min

treatment (Figure 5A, lanes 4 and 7). Prostratin-induced NF-kB

DNA-binding activity was weaker and had a slower kinetics than

that induced by TNFa (Figure 5A, compare lane 3 with lane 2), in

agreement with a previous report [28]. Supershift assays indicated

that the observed retarded complex corresponded to the p50/p65

heterodimer (data not shown). When prostratin was combined

with VPA, an induction of NF-kB binding activity stronger than

that obtained with prostratin alone was observed at the two time

points tested (Figure 5A, compare lane 5 with lane 3, and lane 8

with lane 6). Noteworthy, whereas prostratin-induced NF-kB

DNA-binding activity faded away after a 90 min-treatment, VPA

prolonged this activity (Figure 5A, compare lane 6 with lane 8).

NF-kB activation requires signal-coupled phosphorylation and

degradation of the inhibitors of kB (IkBs, including IkBa), which

bind and sequester NF-kB dimers in an inactive form in the

cytoplasm of resting cells [55]. In order to determine whether the

increased nuclear NF-kB DNA-binding activity observed in

response to prostratin+VPA versus prostratin treatment resulted

from an increased degradation of cytoplasmic IkBa, we monitored

the presence of IkBa as a function of time in the cytoplasm after

treatment with TNFa, prostratin, VPA or prostratin+VPA. TNFainduced a strong IkBa degradation after a 10 min treatment

(Figure 5B, lane 2), whereas VPA alone had little effect on IkBacytoplasmic levels (Figure 5B, lanes 4 and 7). A 30 min-treatment

with prostratin induced IkBa degradation to a similar extent than

a 10-min treatment with TNFa (Figure 5B, compare lane 3 with

lane 2), thus confirming that prostratin activate the NF-kB

pathway with a delayed kinetics compared to TNFa. Remarkably,

whereas prostratin-induced IkBa degradation was followed by its

reappearance after a 1 h treatment (Figure 5B, lane 6), complete

reappearance of cytoplasmic IkBa at this time point was not

detected after the combined treatment prostratin+VPA (Figure 5B,

lane 8).

Our results demonstrated that VPA prolonged and increased

prostratin-induced NF-kB DNA-binding activity and that this

correlated with a prolonged IkBa degradation. This could explain,

at least in part, the transcriptional synergism we observed between

prostratin and VPA on the HIV-1 promoter.

The combined treatment prostratin+VPA causes a morerapid and pronounced nucleosomal remodeling than thecompounds alone

We have previously reported that a single nucleosome (nuc-1)

located immediately downstream of the HIV-1 transcription start

site is remodeled during transcriptional activation of the HIV-1

promoter and that this remodeling is necessary for viral

transcription [4,5,56]. To examine the effects of prostratin+VPA

on nuc-1 remodeling, we determined the ability of the restriction

endonuclease AflII, cutting within nuc-1 DNA, to cut genomic U1

DNA in the context of intact nuclei. The enzyme was incubated

with purified nuclei from U1 cells mock-treated or treated as

indicated in Figure 6B. DNA was purified and digested in vitro with

PstI and analyzed by Southern blotting using indirect end-labeling.

At each time point tested, the combined treatment prostra-

tin+VPA provoked an increase in nuc-1 DNA accessibility more

pronounced than the increases observed after treatment with each

drug individually (Figure 6B, compare lane 4 with lanes 2 and 3,

lane 7 with lanes 5 and 6, and lane 10 with lanes 8 and 9). Of note,

the 2 h combined treatment resulted in nuc-1 remodeling to the

same extent as the control TNFa treatment at 10 min (Figure 6B,

compare lane 10 with lane 11). Similar results were obtained with

HinfI, another restriction endonuclease cutting within nuc-1 DNA

Figure 4. Effect of prostratin+VPA on transcriptional activity of LTRs from several HIV-1 major (M) group subtypes. Jurkat cells weretransiently transfected with LTR-luciferase reporter constructs (LTRs from subtypes A, B, C1, D, CRF01_AE, F, G, CRF02_AG). At 20 h posttransfection,transfected cells were mock-treated or treated with prostratin (5 mM), VPA (2.5 mM), or prostratin+VPA. At 42 h posttransfection, cells were lysed andassayed for luciferase activity. Luciferase activities derived from the HIV-1 LTRs were normalized with respect to protein concentration using theDetergent-Compatible Protein Assay. Results are presented as histograms indicating inductions by the compounds (in fold) with respect to the basalactivity of each pLTR-luc construct, which was assigned a value of 1. Each value is the mean6SE from two separate experiments performed intriplicate.doi:10.1371/journal.pone.0006093.g004



and at two additional sites, thereby generating three bands (named

x, y and z; Figure S4A and S4B) on Southern blots under

incomplete digestion. The accessibility of these two additional sites

(Figure S4B, bands y and z) remained unaffected by the different

treatments.

Figure 5. Effect of VPA on prostratin-mediated NF-kB signalingpathway activation. Nuclear (A) or cytoplasmic (B) extracts wereprepared from Jurkat cells mock-treated or treated with TNFa (10 ng/ml), prostratin (5 mM), VPA (2.5 mM) or with prostratin+VPA for differenttime periods. A. An oligonucleotide corresponding to the HIV-1 LTR NF-kB sites was used as probe in EMSAs with the nuclear extracts. Ascontrol for equal loading, the bottom panel shows comparability of thevarious nuclear extracts assessed by EMSA with an Oct-1 consensusprobe. An experiment representative of three independent experimentsis shown. B. Cytoplasmic extracts were analyzed by Western blottingusing an anti-IkBa antibody (top panel) and an anti-actin antibody (datanot shown) as internal control. Levels of IkBa and actin were quantifiedby chemiluminescence analysis using ChemiDoc XRS System (Biorad)and results are expressed as IkBa amount/actin amount (bottom panel).The ratio obtained with the mock-treated Jurkat cells was arbitrarilyassigned a value of 100%. An experiment representative of twoindependent experiments is shown.doi:10.1371/journal.pone.0006093.g005

Figure 6. Remodeling of nuc-1 after prostratin+VPA versusVPA treatment. (A) Diagram indicating the positions of nucleosomesin the 59 portion of the HIV-1 genome, the HinfI and AflII (*) cutting sitesand the probe used in indirect end-labeling. An asterisk, representingthe AflII cutting site, is located next to the band on the gel to permit itsidentification. (B) Nuclei were prepared from U1 cells mock-treated ortreated with TNFa (10 ng/ml) (10 min), prostratin (5 mM), VPA (2.5 mM)and prostratin+VPA for 30 min, 1 h or 2 h and digested with AflII. AfterDNA purification and in vitro restriction with PstI, DNA samples wereanalyzed by indirect end-labeling using probe A (top panel) [93]. Sizemarkers (a, b, c, d, e, f, g) have been previously described [93].Quantification of the PstI-AflII bands was performed by radioimaginganalysis using an InstantImager (Packard) (bottom panel) and results arepresented as histograms indicating the band intensities relative to theintensity observed with mock-treated U1 cells, which was arbitrarilyassigned a value of 1. Each value is the mean6SE of three separateexperiments performed in duplicate.doi:10.1371/journal.pone.0006093.g006



In order to further examine, at the chromatin level, the

molecular mechanisms involved in the prostratin+VPA synergism,

we analyzed levels of histone H4 acetylation (an activation mark)

in the nuc-1 region. To this end, we performed chromatin

immunoprecipitation (ChIP) assays using U1 cells mock-treated or

treated for different time periods with prostratin and VPA alone or

in combination (Figure S5 and Text S3). VPA induced nuc-1

histone H4 acetylation, a result in agreement with previous reports

[14,57]. Prostratin slightly and gradually induced histone H4

acetylation starting at the 20 min treatment up to the 2 h

treatment. This is in agreement with a previous study demon-

strating that during HIV-1 transcriptional activation by the

phorbol ester PMA, histone acetyltransferases are recruited to

the viral promoter via NF-kB [58]. Importantly, our results

indicated that, at each time point tested, the combined treatment

prostratin+VPA did not induce a level of acetylated histone H4

higher than the level observed after treatments with VPA alone

(Figure S5), suggesting that the more pronounced nuc-1

remodeling we observed by indirect end-labeling following

combined versus individual treatment was not due to a cooperative

induction of histone H4 acetylation.

These results demonstrated that the combined treatment

prostratin+VPA resulted in a more rapid and pronounced nuc-1

remodeling than the treatments with the compounds alone.

The combined treatment prostratin+VPA synergisticallyinduces transcription from the viral LTR

In order to test the effect of prostratin+VPA treatment on

transcriptional activation of the HIV-1 promoter, we first

monitored the RNA polymerase II (RNAPII) abundance in the

5’LTR region. To this end, we performed ChIP assays in U1 cells

after treatment with prostratin alone, VPA alone or prostra-

tin+VPA (Figure 7A). We used a primer pair overlapping the 39

extremity of the LTR U5 region and the 59 extremity of the leader

region allowing to specifically determine RNAPII occupancy at

the 5’LTR. After a 2 h combined treatment, we observed a

cooperative effect of prostratin and VPA on RNAPII recruitment

to the HIV-1 promoter and thus likely on transcription (Figure 7A).

To explore the functional relevance of this cooperative

recruitment, we quantified initiated and elongated HIV-1

transcripts in U1 cells mock-treated or treated with prostratin,

VPA or combination of both for different time periods (1 h, 2 h,

4 h). Initiated versus elongated HIV-1 transcripts were measured

by quantitative real-time RT-PCR with primer sets targeting four

different proviral regions: 1) TAR in the 59 60 bp; 2) pol gene

located roughly 2.9 kb downstream of the 5’LTR; 3) vif gene

located roughly 4.4 kb downstream of the 5’LTR; 4) nef gene,

located roughly 8.3 kb downstream of the HIV-1 LTR. Theses

primer sets allow analysis of transcriptional initiation and

elongation at progressive positions throughout the HIV-1 provirus.

As shown in Figure 7B, after a 4 h treatment with prostratin and

VPA alone, we observed increased relative amounts of both

initiated and elongated transcripts. Importantly, at the same time

point, the prostratin+VPA combined treatment caused a syner-

gistic accumulation of initiated and elongated transcripts

(Figure 7B). Indeed, the prostratin+VPA cotreatment induced a

7.8-fold increase in initiated transcripts and 8.3-fold (pol probe),

7.0-fold (vif probe) and 5.6-fold (nef probe) increases in elongated

transcripts compared to the mock-treated cells.

Altogether, our results measuring the RNAPII recruitment to

the HIV-1 promoter and the amount of transcripts demonstrated

that the prostratin+VPA synergism took place, at least in part, at

the transcriptional initiation and elongation levels and demon-

strated a temporal correlation between the degree of nuc-1

remodeling and the level of HIV-1 transcription.

Prostratin and HDACIs induce HIV-1 recovery in CD8+-depleted PBMCs and in HLA DR2 CD4+ T cells from HIV-1positive individuals with undetectable viral load

Our results suggested that a combination of prostratin+HDACI

(such as VPA, SAHA or NaBut) could have a higher potential than

the compounds alone in reactivating HIV-1 expression from cells

isolated from HIV-1-infected individuals with undetectable viral

load (,50 copies HIV-1 RNA/ml of plasma). To evaluate this

hypothesis, we purified PBMCs from blood of 52 selected patients

(Materials and Methods) and depleted them for CD8+ T cells,

CD8+ T cells having an antiviral activity [59]. Importantly, these

purified cells were cultured both in the absence of IL-2 and in the

absence of allogenic stimulation (i.e. coculture with PBMCs from

an uninfected individual) to avoid extensive nonspecific T-cell

activation and proliferation, and thus resulting in no amplification

of the genomic viral RNA level. After a one-day culture, cells were

mock-treated or treated with anti-CD2+anti-CD28 antibodies (as

a positive control), prostratin, HDACIs (VPA or SAHA) or with

combinations of prostratin+HDACIs. Six days after treatment, we

measured HIV-1 genomic RNA concentrations in culture

supernatants.

For 10 out of 52 CD8+-depleted PBMC cultures, we detected

viral RNA in the supernatant even in the absence of any

treatment. Therefore, these data were removed from our study.

Characteristics of the 42 remaining patients (named X1 to X42)

are presented in Table 1. This table also indicates, for each PBMC

culture, the combination of prostratin+HDACI tested (prostra-

tin+VPA and/or prostratin+SAHA) and the levels of rescue

obtained after anti-CD2+anti-CD28 treatment. These levels of

rescue showed that the 17 CD8+-depleted PBMC cultures,

presenting no viral outgrowth following treatment with prostratin

and HDACI(s) individually or in combination, exhibited no or

very weak levels of genomic viral RNA after anti-CD2+anti-CD28

costimulation compared to the levels observed with the PBMC

cultures presenting viral reactivation after drug treatments

(Table 1, compare patients X1 to X25 with X26 to X42). For

25 of the 42 PBMC cultures, we detected HIV-1 reactivation after

prostratin, VPA, SAHA and/or prostratin+HDACI exposure

(Tables 1 and 2, patients X1 to X25). We did not observe any

significant correlation between age, CD4 counts or length of

undetectable viremia, and the potency of our compounds to

reactivate HIV-1 from latency. In 17 of these 25 cultures,

prostratin alone reactivated viral expression, thereby confirming

its great potential as an HIV-1 replication inducer (Table 2, X1,

X2, X4 to X6, X9, X11, X14, X16, X18 to X25). VPA and

SAHA alone reactivated viral expression in only 3 cell cultures,

(Table 2, X4, X11, X14 and X4, X23, X24, respectively). In 8

cultures (Table 2, X3, X7, X8, X10, X12, X13, X15, X17), no

drug used alone could reactivate viral expression.

In 17 out of the 25 reactivated cultures, HIV-1 replication was

synergistically induced by a combination prostratin+HDACI

(Table 2, patients X1 to X17). These 17 CD8+-depleted PBMC

cultures can be subdivided in three categories for each

combination prostratin+HDACI (Figure 8A, 8B, 8C for prostra-

tin+VPA and Figure 8D, 8E, 8F for prostratin+SAHA). The first

category concerned 3 cultures for the combination prostra-

tin+VPA (Figure 8A, X4, X11, X14) and one culture for the

combination prostratin+SAHA (Figure 8D, X4). After treatment

with prostratin, VPA or SAHA alone, we detected HIV-1 genomic

RNA in the supernatants. After combined treatments with

prostratin+VPA or prostratin+SAHA, we measured a synergistic



increase in viral RNA copy number. The second category

concerned CD8+-depleted PBMC cultures in which we detected

HIV-1 reactivation after treatment with prostratin, prostra-

tin+VPA (Figure 8B, X1, X2, X5, X6, X9) or prostratin+SAHA

(Figure 8E, X9, X11, X14, X16) but not after treatment with VPA

or SAHA alone. In the third category, we grouped the results

obtained with CD8+-depleted PBMC cultures in which we detect

viral RNA only after a combined treatment with prostratin+VPA

(Figure 8C, X10, X12, X13) or prostratin+SAHA (Figure 8F, X3,

X7, X8, X10, X12, X13, X15, X17). In cultures from patients

X18 to X25, no synergistic effect of the drug combinations was

observed (Table 2).

Figure 7. Effects of prostratin+VPA on HIV-1 transcription. (A) RNAPII occupancy of the HIV-1 promoter region was assessed by ChIPexperiments. U1 cells were mock-treated or treated with prostratin (5 mM), VPA (2.5 mM) and prostratin+VPA for 30 min, 1 h or 2 h. The proteinswere cross-linked with formaldehyde for 10 min and DNA sheared. The cross-linked protein/DNA complexes were immunoprecipitated with an anti-RNAPII antibody. The protein/DNA cross-links were reversed and the purified DNA was amplified and quantified by real-time PCR using primersamplifying the region overlapping the LTR U5 region and the primer binding site. Values were normalized to those obtained with the 18S rRNAprimers and expressed as fold inductions relative to the value measured in mock-treated U1 cells, which was arbitrarily set at a value of 1. Each valueis the mean6SE from three separate experiments performed in triplicate. (B) Measurement of initiated and elongated HIV-1 transcripts followingdrug treatments. Total RNA was extracted from U1 cells mock-treated or treated with prostratin (5 mM), VPA (2.5 mM) and prostratin+VPA for 1 h, 2 hor 4 h. Initiated (primers tar) or elongated (primers pol, vif and nef) transcripts were quantified by quantitative real-time RT-PCR. Values werenormalized to those obtained with the b-actin primers and expressed for each primer pair as fold inductions relative to the values measured in mock-treated U1 cells, which were arbitrarily set at a value of 1. The results shown are the average of eight independent quantitative real-time PCRs fromfour separate reverse transcriptions from two independent cell culture treatments. The error bars show the standard errors. An experimentrepresentative of two independent experiments is shown.doi:10.1371/journal.pone.0006093.g007



Table 1. Characteristics of HIV-1-infected individuals named X1 to X42.

PatientsAge(years)

CD4 count atthe time ofthe study(cells/ml)

Antiviral therapyat the time ofthe studya

Durationof therapy(years)

Duration withundetectableplasma HIV-1RNA level (,50copies/ml ofplasma) (years) Reactivationb

HIV-1 RNA levelafter treatmentwith anti-CD2+anti-CD28antibodiesc

(copies/ml)

Combination ofprostratin+HDACItested

X1 49 441 CBV, NVP 5 5 + 4070 Pro+VPA

X2 52 1000 ATV, EFV, RTV, TDF 8 3 + 42000 Pro+VPA

X3 56 596 CBV, DDI, fAPV 8 4 + 3920 Pro+VPA/Pro+SAHA

X4 45 1064 3TC, fAPV, RTV, TDF 13 2 + 39800 Pro+VPA/Pro+SAHA

X5 29 479 3TC, DDI, EFV 2 2 + 1253 Pro+VPA

X6 67 717 CBV, EFV 12 4 + 52300 Pro+VPA

X7 52 724 TZV 10 8 + 98000 Pro+VPA/Pro+SAHA

X8 45 542 3TC, EFV, fAPV, RTV, TDF 10 3 + 232 Pro+VPA/Pro+SAHA

X9 35 962 RTV, fAPV, CBV 12 1 + 467 Pro+VPA/Pro+SAHA


X11 50 786 TDF, FTC, EFV 2 2 + 6665 Pro+VPA/Pro+SAHA


X13 38 1239 NVP, TDF, 3TC 9 8 + 219 Pro+VPA/Pro+SAHA

X14 66 1073 TDF, 3TC, RTV, ATV 10 1 + 5053 Pro+VPA/Pro+SAHA

X15 50 1748 CBV, NVP 11 10 + 248 Pro+SAHA

X16 40 722 RTV, fAPV, TDF, FTC 2 2 + 72 Pro+SAHA

X17 42 614 3TC, TDF, NVP 4 3 + 105 Pro+SAHA

X18 66 842 3TC, ABC, EFV 4 1 + 172 Pro+VPA

X19 57 587 CBV, EFV, LPV/r 8 3 + 3154 Pro+VPA

X20 62 437 D4T, KVX, NVP 13 7 + 11200 Pro+VPA/Pro+SAHA

X21 52 934 3TC, ABC, fAPV, RTV 8 4 + 6478 Pro+VPA

X22 43 680 NVP, fAPV, KVX, RTV 12 6 + 15394 Pro+VPA/Pro+SAHA

X23 30 536 RTV, fAPV, FTC, TDF 2 2 + 611 Pro+VPA/Pro+SAHA

X24 66 1183 ATV, 3TC, TDF 13 1 + 3121 Pro+SAHA

X25 34 721 3TC, TDF, NVP 11 10 + 13465 Pro+SAHA

X26 29 617 3TC, ABC, LPV/r 1 1 - 0 Pro+VPA

X27 54 689 3TC, TDF 9 2 - 0 Pro+VPA

X28 36 774 CBV, NVP 4 4 - 0 Pro+VPA

X29 62 611 TZV 8 2 - 99 Pro+VPA

X30 61 502 LPV/r, NVP 6 2 - 360 Pro+VPA

X31 55 959 TZV 5 5 - 0 Pro+VPA/Pro+SAHA

X32 27 501 3TC, ABC, EFV 3 3 - 0 Pro+VPA

X33 61 508 EFV, KVX, TDF 9 6 - 102 Pro+VPA

X34 46 515 TZV 9 9 - 0 Pro+VPA

X35 58 490 CBV, NVP 8 3 - 142 Pro+VPA/Pro+SAHA

X36 51 682 IDV, NVP, RTV 10 5 - 463 Pro+VPA/Pro+SAHA

X37 34 599 CBV, fAPV, RTV 6 6 - 299 Pro+VPA

X38 44 1048 AZT, DDI 11 10 - 0 Pro+VPA/Pro+SAHA

X39 38 857 FTC, TDF, EFV 5 4 - 0 Pro+SAHA

X40 40 798 EFV, KVX 4 2 - 0 Pro+SAHA

X41 60 697 RTV, ATV, CBV 11 5 - 1241 Pro+SAHA

X42 43 643 NVP, 3TC, TDF 5 5 - 0 Pro+SAHA

Mean 47.7 746 7.6 4.2

Range 27–67 437–1748 1–13 1–10

aNucleoside analogs: Abacavir (ABC), Didanosine (DDI), Stavudine (D4T), Lamivudine (3TC), Zidovudine/Lamivudine (CBV), Zidovudine/Lamivudine/Abacavir (TZV),Tenofovir (TDF), Abacavir/Lamivudine (KVX), Emtricitabine (FTC), Zidovudine (AZT). Nonnucleoside reverse transcriptase inhibitors: Nevirapine (NVP), Efavirenz (EFV).Protease inhibitors: Indinavir (IDV), Ritonavir (RTV), Lopinavir/Ritonavir (LPV/r), Atazanavir (ATV), Fosamprenavir (fAPV).

bReactivation of HIV-1 expression (+).clevel of HIV-1 expression were determined by measuring HIV-1 RNA levels as described in Materials and Methods.doi:10.1371/journal.pone.0006093.t001



For the CD8+-depleted PBMC cultures from patients X26 to

X42, we did not detect any viral outgrowth following treatment

with prostratin and HDACIs individually or in combination

(Table 1). However, we cannot exclude that, in some of these

cultures, reactivation levels were under the detection limit of the

Roche Amplicor assay.

Among the cell types present in CD8+-depleted PBMCs,

latently-infected resting memory CD4+ T cells that harbor

integrated replication-competent viral DNA represent the primary

long-lived source of HAART-persistent HIV-1 [13,25,60]. These

resting cells carrying a non-productive HIV-1 infection, derived

from infected CD4+ lymphoblasts that have reverted back to a

resting memory state and show a specific pattern of surface

markers (including CD4+and HLA DR2) [13]. To evaluate in

HLA DR2 CD4+ T cells the results we obtained in CD8+-depleted

PBMCs, we purified HLA DR2 CD4+ T cells from blood samples

obtained from 9 HAART-treated patients selected as described in

Materials and Methods. Characteristics of patients named X43 to

X51 are presented in Table 3. Because of limited amounts of HLA

DR2 CD4+ T cells, we tested the reactivation effect of the

combined treatment prostratin+SAHA, shown here above to

reactivate HIV-1 expression with higher efficiency than the

combination prostratin+VPA in CD8+-depleted PBMC cultures.

We demonstrated that HIV-1 replication was reactivated in 7

HLA DR2 T-cell cultures out of 9 tested, with RNA viral loads

ranging from 215 to 6684 copies/ml depending on the patient

culture (Figure 9).

In our studies, we used 66106 CD8+-depleted PBMCs or 26106

HLA DR2 CD4+ T cells per well, therefore, there are probably

very few latent cells in each well. It is possible that, in some cases,

the differences in the HIV-1 recovery levels observed in the

different cultures as well as the failure of the drugs alone or in

combination to stimulate HIV-1 in some cultures may be due to

partioning of the few infected cells. However, given the high

number of CD8+-depleted PBMC cultures we tested (42) and the

results obtained in several latently-infected cell lines, it seems

unlikely that the results reported here would be due to random

distributions of the infected cells between culture wells.

In conclusion, although the HIV-1 reactivation levels in

response to the different treatments varied importantly from one

patient to the other, the clinically used HDACIs (VPA or SAHA)

alone or the non-tumor-promoting phorbol ester prostratin alone

induced HIV-1 outgrowth in CD8+-depleted PBMC cultures in

the absence of added IL-2 or of allogenic stimulation. Importantly,

the combination prostratin+HDACI allowed HIV-1 reactivation

in a higher number of cell cultures compared to the number of

positive cultures observed with the activators individually, and to a

higher extent than these activators alone. Moreover, the combined

treatment prostratin+SAHA efficiently reactivated viral replication

in resting CD4+ T cells, which constitute the most stable HIV-1

reservoir.

Discussion

The HIV-1 latent reservoirs decrease only slowly in patients

undergoing HAART. It is estimated that decades of treatment

would be required to completely eliminate the latent virus. Ideal

compounds for viral purging would induce viral replication in a

wide variety of infected cell types without causing massive T-cell

activation and proliferation to prevent the generation of new target

cells for the neosynthetized virus, while maintaining the patient on

HAART to prevent a spreading infection. In this report, we

combined two different types of compounds that meet these

criteria: the non-tumor-promoting NF-kB inducer prostratin and

HDACIs already in use for other diseases and thus clinically

promising for HIV-1 flushing strategies. We demonstrated a

synergistic activation of HIV-1 production by combinations of

prostratin+HDACI in the latently-infected monocytic cell line U1

and T-lymphoid J-Lat 8.4 and 15.4 cell lines. In these later cell

lines, we showed that the synergistic effect was due, at least

partially, to the recruitment of unresponsive cells into the

expressing cell population. VPA increased prostratin-induced

NF-kB activation and potentiated nuc-1 remodeling. The

prostratin+VPA combined treatment caused a synergistic accu-

mulation of initiated and elongated transcripts, as demonstrated by

quantitative real-time RT-PCR. We performed viral reactivation

assays in cell cultures prepared from HAART-treated HIV-1-

infected individuals with undetectable viral load and showed that a

combination prostratin+VPA or prostratin+SAHA induced HIV-1

recovery in 25 of the 42 CD8+-depleted PBMC cultures tested

(with a synergistic recovery observed in 17 out of these 25).

Moreover, the combination prostratin+SAHA reactivated viral

Table 2. Reactivation profiles observed in CD8+-depletedPBMC cultures from patients X1 to X25.

Treatments Pro VPA Pro+VPA SAHA Pro+SAHA

Patients

X1 + 2 +* / /

X2 + 2 +* / /

X3 2 2 2 2 +*

X4 + + +* + +*

X5 + 2 +* / /

X6 + 2 +* / /

X7 2 2 2 2 +*

X8 2 2 2 2 +*

X9 + 2 +* 2 +*

X10 2 2 +* 2 +*

X11 + + +* 2 +*

X12 2 2 +* 2 +*

X13 2 2 +* 2 +*

X14 + + +* 2 +*

X15 2 / / 2 +*

X16 + / / 2 +*

X17 2 / / 2 +*

X18 + 2 + / /

X19 + 2 + / /

X20 + 2 + 2 +

X21 + 2 + / /

X22 + 2 + 2 +

X23 + 2 + + +

X24 + / / + +

X25 + / / 2 +

Percentage ofreactivation

68% 15% 85% 17% 100%

Cultures were mock-treated or treated with prostratin (5 mM), VPA (2.5 mM) orSAHA (2.5 mM) alone or in combination. Reactivation of HIV-1 expression (+),synergistic viral reactivation (+*) or no viral outgrowth (2) were determined bymeasuring HIV-1 RNA levels as described in Materials and Methods. For somepatients, insufficient number of CD8+-depleted PBMCs was available for testingthe considered conditions (/).doi:10.1371/journal.pone.0006093.t002



replication in 7 of the 9 HLA DR2 CD4+ T cell cultures prepared

from additional patients. This study suggests that this type of

combinatory activation approach could be used as an inducible

adjuvant therapy with efficient HAART to accelerate latent HIV-

1 clearance.

Viral reactivation trials with activating agents such as IL-2 and

OKT3 (a murine monoclonal anti-CD3 antibody), have shown

little, if any, reduction in viral rebound after cessation of HAART

[60] and their in vivo application has resulted in deleterious side

effects [61]. In 2002, we proposed the use of HDACIs as an

inductive adjuvant therapy with efficient HAART aimed at

forcing HIV-1 gene expression in the latently-infected cells [3,10].

Here, we demonstrated that the 11 inhibitors of class I and II

HDACs (but not the two inhibitors of class III HDACs we tested)

all induced HIV-1 production in U1 cells, independently of the

structural group these HDACIs belonged to. This is in agreement

with data from several groups reporting the recruitment of class I

HDACs to the HIV-1 promoter [62,63,64,65,66].

Figure 8. Prostratin+HDACIs synergistically induce HIV-1 recovery in CD8+-depleted PBMCs from HAART-treated patients withundetectable viral load. CD8+-depleted PBMCs from patients X1 to X17 were mock-treated or treated with prostratin (5 mM), VPA (2.5 mM) (panelsA, B and C), SAHA (2.5 mM) (panels D, E and F) alone or in combination. Six days after treatment, HIV-1 genomic RNA concentrations were measuredin culture supernatants as described in Materials and Methods. When a single patient PBMCs culture presented distinct profiles depending on thetreatment (prostratin+VPA or prostratin+SAHA), this culture was shown in several categories.doi:10.1371/journal.pone.0006093.g008



Because VPA is in wide use to treat common chronic

neurological and psychiatric disorders and often given simulta-

neously to HAART for long periods of time to HIV-1-infected

individuals, many recent studies aimed at determining VPA

potential to accelerate clearance of HIV-1. Whereas two works

have suggested that the VPA could be a good candidate to deplete

latent infection [14,15], more recent studies did not confirm these

results [18,19,20,67] (and this paper). In agreement with this, our

reactivation assays showed that VPA alone induced HIV-1

recovery in only 3 out of 33 CD8+-depleted PBMC cultures

prepared from patients receiving HAART. However, our results

indicated that VPA used in combination with prostratin was more

efficient in reactivating HIV-1 replication than these compounds

used individually. Indeed, in some cell cultures in which VPA

alone did not cause any detectable HIV-1 reactivation, this

HDACI potentiated prostratin-induced HIV-1 recovery. More-

over, in the 3 CD8+-depleted PBMC cultures in which VPA alone

reactivated viral expression, we detected a synergistic recovery of

HIV-1 RNA production after the combined treatment prostra-

tin+VPA. Therefore, combined with other kinds of HIV-1

inducers, VPA could have an impact on the decay of latent

reservoirs, despite its weak HDAC inhibitor activity. Its combined

administration could be even more beneficial for long-term

purging therapies than other FDA (Food and Drug Administra-

tion)-approved more potent HDAC inhibitors, such as SAHA that

exhibits relative toxicities (diarrhea, fatigue, nausea, anorexia and

dehydratation) [42,49]. Such combination approaches could

provide an important HIV-1 activation leading to a decline of

HIV-1 reservoirs level sufficient to allow an efficient control of the

infection by the host immune system, and might thus allow

therapeutic interruptions (‘‘treatment-free windows’’).

Our FACS analyses of the J-Lat clones revealed that the

proportion of J-Lat cells displaying GFP epifluorescence was

synergistically increased by prostratin+HDACI cotreatments

compared to treatments with the compounds alone. This implied

that the synergistic effect observed following a prostratin+HDACI

cotreatment on viral p24 antigen production was due, at least in

part, to the recruitment of unresponsive cells into the responsive

cell population. However, we cannot exclude that part of this

synergistic effect also resulted from a higher level of expression

from the already responding cell population. The cells containing

HIV-1 promoters, that were reactivable neither by HDACI alone

nor by prostratin alone but were reactivable by the combination,

are likely further locked in an epigenetic silent state compared to

the more easily reactivable promoters, which are responsive to a

drug alone. Those J-Lat cells that remain unresponsive to the

combined treatment are probably regulated by repressive

epigenetic marks that cannot be reversed solely by inducing NF-

kB and inhibiting HDAC activity.

Among the elements involved in HIV-1 reactivation, NF-kB

activity is of paramount importance. Indeed, the two NF-kB-

binding sites in the enhancer are critical for LTR promoter activity

and important for optimal HIV-1 replication [11]. Here, we report

that prostratin alone induced IkBa degradation and NF-kB DNA-

binding activity, although the prostratin-induced activation of the

NF-kB signaling pathway was weaker in intensity and had a

Figure 9. Effects of prostratin+SAHA treatment on HIV-1recovery in HLA DR2 CD4+ T cells. HLA DR2 CD4+ T cells fromHAART-treated patients with undetectable viral load (X43 to X51) weremock-treated or treated with the combination prostratin (5 mM)+SAHA(2.5 mM). Six days after treatment, HIV-1 genomic RNA concentrationswere measured in culture supernatants as described in Materials andMethods.doi:10.1371/journal.pone.0006093.g009

Table 3. Characteristics of HIV-1-infected individuals named X43 to X51.

Patients Age (years)CD4 count at the timeof the study (cells/ml)

Antiviral therapy at thetime of the studya

Duration oftherapy (years)

Duration with undetectableplasma HIV-1 RNA level (,50copies/ml of plasma) (years)

X43 47 325 TVD, RTV, ATV 9 2

X44 54 307 TVD, EFV 14 5

X45 49 1298 LPV/r, EFV, TVD 18 7

X46 37 461 TVD, EFV 8 7

X47 49 759 TVD, SQV, RTV 8 6

X48 41 707 EFV, LPV/r, FTC 13 7

X49 35 463 KVX, NVP 8 8

X50 35 841 LPV/r, TVD 2 2

X51 60 608 CBV, NVP 16 9

Mean 45.2 641 10.7 5.9

Range 35–60 307–1298 2–18 2–9

aNucleoside analogs: Tenofovir/Emtricitabine (TVD), Emtricitabine (FTC), Zidovudine/Lamivudine (CBV), Abacavir/Lamivudine (KVX). Nonnucleoside reverse transcriptaseinhibitors: Nevirapine (NVP), Efavirenz (EFV). Protease inhibitors: Ritonavir (RTV), Lopinavir/Ritonavir (LPV/r), Atazanavir (ATV), Saquinavir (SQV).

doi:10.1371/journal.pone.0006093.t003



delayed kinetics compared to that elicited by TNFa, in agreement

with a previous study [28]. Importantly, we showed that VPA

increased and prolonged prostratin-induced NF-kB DNA-binding

activity. This is consistent with the fact that acetylation/

deacetylation events regulate the NF-kB activating signaling

pathway at multiple levels, including the direct acetylation of

NF-kB [55]. Indeed, Lys221 acetylation enhances p65 DNA-

binding activity and, together with acetylation at Lys218, impairs

its assembly with IkBa, thereby impeding IkBa-dependent nuclear

export of the NF-kB complex and enabling prolongation of the

NF-kB response [55]. Moreover, the acetylated form of p50 binds

target sequences with higher affinity than the unacetylated form

does [55]. Noteworthy, the prolonged NF-kB DNA-binding

activity that we observed here was correlated with a prolonged

IkBa degradation. IkBa degradation is dependent upon its

phosphorylation by the IkB kinase (IKK) complex and its

subsequent ubiquitination. Thereby, VPA could prolong the

prostratin-induced IkBa degradation by prolonging the IKK

activity, as we have previously shown for the TNFa+TSA

combined treatment [21].

The nucleosome nuc-1 is positioned immediately downstream

of the HIV-1 transcription start and is remodeled upon

transcriptional activation by Tat and the HDACIs TSA or

Trapoxin in the absence of NF-kB stimulation [4,5]. These

observations suggest that nuc-1 plays a repressive role in HIV-1

transcription and that histone acetylation could be involved in

overriding this effect. Accordingly, we reported here that the

HDACI VPA induced nuc-1 histone H4 acetylation. Importantly,

we showed that the combination prostratin+VPA did not induce a

level of acetylated histone H4 higher than the level observed with

VPA alone. Therefore, the cooperative nuc-1 remodeling we

observed by indirect end-labeling following a prostratin+VPA

cotreatment was not due to a cooperative induction of histone H4

acetylation. Chromatin structure can be altered both by histone

posttranslational modifications (such as acetylation), which alter

histone/DNA or histone/histone interactions, and by ATP-

dependent chromatin remodeling complexes (such as SWI/SNF)

[68]. It has been previously reported that these SWI/SNF

complexes are recruited at gene promoters by transcription

factors, including NF-kB [69,70] and AP-1 [71,72], or by

acetylated histones [73]. Therefore, by inducing NF-kB and/or

AP-1 binding to the HIV-1 5’LTR [22,28,32], prostratin could be

involved in the remodeling of nuc-1 through the recruitment of

SWI/SNF by these transcription factors. This hypothesis could

explain the more pronounced nuc-1 remodeling we observed in

the postintegration latency model cell line U1 after the

prostratin+VPA cotreatment compared to the treatments by the

drugs alone.

We demonstrated a temporal correlation between the degree of

nuc-1 remodeling and the level of transcription in U1 cells by

showing that the prostratin+HDACI-induced increase in chroma-

tin remodeling at 2 h postinduction was correlated with an

increased RNAPII recruitment to the viral LTR at the same time

point and to an increased accumulation of both initiated and

elongated transcripts at 4 h postinduction. Expression of full-

length HIV-1 transcripts requires the action of the cellular kinase

P-TEFb, composed of a catalytic subunit, cyclin-dependent kinase

9 (Cdk9) and a regulatory subunit, cyclinT1. The absence or

inactivity of this protein, while generating short viral transcripts,

fails to support effective viral replication. P-TEFb mediates

transcriptional elongation of the bound polymerase complexes

by phosphorylating the C-terminal domain (CTD) of RNAPII.

Although P-TEFb is mainly recruited via the viral transactivator

Tat, other mechanisms that can ensure P-TEFb recruitment and

thereby transcriptional elongation have been described and can

account for signal-induced transcription elongation in Tat-

defective HIV-1 infected cells, such as the U1 cell line. Notably,

Sp1, p65 and histone acetylation can contribute to P-TEFb

recruitment to the HIV-1 promoter [74,75,76,77], via Brd4, a

positive regulatory component of P-TEFb, for p65 and histone

acetylation [75,76,77]. Brd4 is a bromodomain-containing protein

that recognizes acetylated histones [78] as well as acetylated p65

[75]. Altogether, these previous data could explain at the

molecular level our quantitative real-time RT-PCR results

showing that prostratin and VPA synergistically activated

transcriptional elongation in U1 cells. The prostratin+VPA

cotreatment could allow a more important recruitment of the

Brd4/P-TEFb complex to the HIV-1 promoter than the drugs

alone. In addition to activating the NF-kB signaling pathway,

prostratin up-regulates cyclinT1 expression, leading to an

increased association between cyclinT1 and cyclin-dependent

kinase 9 (Cdk9) which results in a more efficient P-TEFb kinase

activity [79].

Our reactivation assays suggested that when combined with

prostratin, SAHA could be a more potent HIV-1 reactivating

agent than VPA. Indeed, whereas a prostratin+VPA cotreatment

reactivated viral expression in 17 out of the 33 CD8+-depleted

PBMC cultures tested (11 presenting synergistic activation), a

combined treatment with prostratin+SAHA reactivated viral

expression in 18 out of 26 CD8+-depleted PBMC cultures (13

presenting synergistic activation) and in 7 out of the 9 HLA DR2

CD4+ T cell cultures we tested. Supporting our data, two very

recent studies, published during the revision of the present paper,

have reported that SAHA reactivates HIV-1 from latently-infected

cells [80,81]. SAHA is administered intravenously to patients with

refractory hematologic and solid tumors and has been approved

for use as a treatment of cutaneous T-cell lymphoma [42,48,49].

Because of its ability to promote HIV-1 expression, especially in

combination with prostratin, SAHA provides additional promise

for the decay of latent HIV-1 infection and deserves further

investigations.

Of note, in 17 out of 42 CD8+-depleted PBMC cultures, we did

not detect any viral outgrowth following treatment with prostratin

and HDACIs individually or in combination and no or very weak

viral outgrowth following an anti-CD2+anti-CD28 treatment

compared to the levels observed with the PBMC cultures

presenting viral reactivation after drug treatments. A possible

explanation to the variegated response to prostratin+HDACI

treatment observed in the CD8+-depleted PBMC cultures could

result from an ‘‘on-off switching’’ of transcriptional competence.

Local chromatin structure at the site of virus integration within the

host genome modulates the level of epigenetic HIV-1 transcrip-

tional repression as well as the ability of the virus to respond to

reactivating stimuli [82]. Indeed, some of the integrated HIV-1

DNA may be under strong epigenetic repression (through DNA

methylation and histone methylation and deacetylation) that

cannot be efficiently reversed by the drugs we used in this study

[7,83]. However, we cannot rule out the possibility that the viruses

present in the samples in which we could not detect any viral

reactivation were defective. Therefore, locus-specific transcrip-

tional repression might account, at least in part, for the

heterogeneous HIV-1 reactivation profiles we observed after

prostratin+HDACI cotreatment.

In conclusion, we report a proof-of-concept study for the

therapeutic potential coadministration of two different kinds of

HIV-1 activators (one acting on the NF-kB pathway and the other

acting on the protein acetylation status) aimed at efficient decay of

latent reservoirs in presence of HAART. However, we did not



observe reactivation of viral replication in all the patient cell

cultures tested, thereby emphasizing the importance to identify

other combinations involving SAHA, prostratin, prostratin-like

compounds with higher potency and/or other kinds HIV-1

inducers (such as inhibitors of histone- and DNA-methyltransfer-

ases). In this regard, a recent study has reported the practical

synthesis in gram quantities and at low cost of prostratin, 12-

deoxyphorbol-13-phenylacetate (DPP) and a variety of new 12-

deoxyphorbol analogs, which might be potentially superior clinical

candidates against latent HIV-1 [34].

Materials and Methods

Cell Lines and Cell CultureThe monocytic cell line U1 [84] and the T-lymphoid cell lines

Jurkat [85], J-Lat 8.4 and J-Lat 15.4 [51] were obtained from the

AIDS Research and Reference Reagent Program (National

Institute of Allergy and Infectious Diseases [NIAID], National

Institute of health [NIH]). All cell lines were grown in RPMI 1640

medium (Gibco-BRL) supplemented with 10% fetal bovine serum,

50 U/ml of penicillin, 50 mg/ml of streptomycin at 37uC in a

humidified 95% air/5% CO2 atmosphere.

ReagentsTNFa (catalog no. 300-01A) was purchased from Immuno-

source. SAHA (suberoylanilide hydroxamic acid) (catalog no. 270-

288-M001) was obtained from Alexis Biochemichals. Prostratin

(12-deoxyphorbol-13-acetate) (catalog no. PE 187-0001), HC-

Toxin (Cyclo(D-prolyl-L-alanyl-D-alanyl-L-2-amino-9,10-epoxy-

8-oxodecanoyl)) (catalog no. GR-320), SBHA (suberoyl bis-

hydroxamic acid) (catalog no. GR-323), Scriptaid (6-(1,3-dioxo-

1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxyhexanamide) (cata-

log no. GR-326) were provided by Tebu-Bio. NaBut (sodium

butyrate) (catalog no. B5887), VPA (2-propylpentanoic acid

sodium or valproic acid) (catalog no. P4543), Depudecin (D-

threo-D-ido-undeco-1,6-dienitol,4,5:8,9-dianhydro-1,2,6,7,11-

pentadeoxy) (catalog no. D5816), apicidin (cyclo[(2S)-2-amino-8-

oxodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-(2R)-2-piperidi-

necarbonyl]) (catalog no. A8851), TSA (trichostatin A) (catalog no.

T8552) were purchased from Sigma-Aldrich. PMA (phorbol

myristate acetate) (catalog no. 524400), Splitomycin (1,2-dihy-

dro-3H-naphtho[2,1-b]pyran-3-one) (catalog no. 567750), MS-

275 (N-(2-Aminophenyl)-4-[N-(pyridine-3-ylmethoxycarbonyl)a-

minomethyl] benzamide) (catalog no. 382147), Sirtinol (2-[(2-

hydroxynaphthalen-1-ylmethylene)amino]-N-(1-phenethyl)benza-

mide) (catalog no. 566320), CBHA (m-carboxycinnamic acid bis-

hydroxamide) (catalog no. 382148) were obtained from Calbio-

chem. All compounds, resuspended and stored as recommended

by the manufacturer, were diluted immediately before use in cell

culture medium.

Virus production assays in U1 and J-Lat 8.4 or 15.4 celllines

HIV-1 production was measured in the U1 and J-Lat 8.4 or

15.4 cell line culture supernatants by determining CA-p24 antigen

concentration by an enzyme-linked immunosorbent assay (In-

nogenetics).

Transient transfection and luciferase reporter assaysJurkat cells were transfected with the pLTR (A, B, C1, D,

CRF01_AE, F, G, CRF02_AG) –luciferase reporter plasmids

(1000 ng) [86] using jetPEITM (POLYplus) according to the

manufacturer’s protocol. Cells were mock-treated or treated with

the different compounds as indicated, lysed and assayed for

luciferase activity as previously described [3].

EMSAsNuclear extracts were prepared and EMSAs with the HIV-1

NF-kB probe were performed as previously described [3]. As

loading controls, the same nuclear extracts were tested for binding

of Oct-1 (octamer-binding protein-1) to an Oct-1 consensus probe

(59-TGTCGAATGCAAATCACTAGAA-39).

Western blot analysesCytoplasmic extracts were prepared as previously described

[87]. Western blots were performed [87] with a rabbit antibody

against IkBa (1/1000 dilution; catalog no. 06-494; Upstate

Biotechnology) or against actin (1/1000 dilution; catalog

no.A2066; Sigma-Aldrich) and a peroxidase-conjugated goat

anti-rabbit IgG (1/3000 dilution; catalog no. 7074; Cell Signaling).

Indirect end-labeling techniqueThe indirect end-labeling experiments were performed as

previously described [4].

Study SubjectsWe selected 52 HIV-1-infected individuals at the St-Pierre

Hospital (Brussels, Belgium) and 9 additional patients at the

Bicetre Hospital (Bicetre, France) on the basis of the following

criteria: all volunteers were treated with HAART for at least 1

year, had an undetectable plasma HIV-1 RNA level (,50 copies/

ml) for at least 1 year and had a level of CD4+ T lymphocytes

higher than 300 cells/mm3 of blood. Characteristics (age, CD4+ T

cell count, antiviral regimens, duration of therapy, duration with

undetectable plasma HIV-1 RNA level) of patients from the St-

Pierre Hospital and from the Bicetre Hospital are presented in

Table 1 and 3, respectively.

Ethics StatementEthical approval was granted by the Human Subject Ethics

Committees of the Saint-Pierre Hospital and of the Bicetre

Hospital. All individuals enrolled in the study provided written

informed consent for donating blood.

Isolation of CD8+-depleted PBMCs and of HLA DR2 CD4+

T lymphocytesCD8+-depleted PBMCs were isolated from fresh whole blood

(50 ml) of the HIV-1-infected individuals described above by

adding RosetteSep human CD8 depletion mixture (StemCell

Technologies) to whole blood samples before density centrifuga-

tion on a Ficoll-Hypaque gradient (Pharmacia). HLA DR2 CD4+

T lymphocytes were isolated as previously described [88]. The

number of cells (CD8+-depleted PBMCs and HLA DR2 CD4+ T

lymphocytes) obtained after isolation varied depending on the

patients. In all cases, cells were washed with RPMI, resuspended at

26106 cells/ml of complete RPMI (RPMI, 10% fetal bovine

serum, supplemented with 50 U/ml of penicillin, and 50 mg/ml of

streptomycin). As a positive control of reactivation, CD8+-depleted

PBMCs were stimulated with anti-CD2+anti-CD8 antibodies used

at saturating concentrations [mAb 39C1.5 (rat IgG2a, anti-CD2)

in combination with mAb 6F10.3 (mouse IgG1, anti-CD2); anti-

CD28 mAbs (clone identification 28.2)] [89,90]. Activation by

anti-CD2+anti-CD28 monoclonal antibodies induces high-level,

long-lasting and monocyte-independent proliferation of resting T

cells, thymocytes and preactivated T cells [91,92].



Roche Amplicor quantitative assessments of HIV-1 RNAOne day after isolation, 66106 CD8+-depleted PBMCs or

26106 HLA DR2 CD4+ T cells were mock-treated or treated with

different compounds. Six days after treatment, culture superna-

tants were tested for quantitative HIV-1 RNA levels by using the

COBAS AmpliPrep/COBAS AMPLICOR HIV-1 MONITOR

Test version 1.5, according to the manufacturer’s instructions

(Roche Diagnostics) (lowest detection limit: 50 copies HIV-1

RNA/ml of plasma).

ChIP assaysChIP assays were performed using the ChIP assay kit (EZ ChIP

technology, Upstate). U1 cells were cross-linked after drug

treatments. To detect chromosomal flanking regions, pellets were

sonicated (Bioruptor sonicator) to obtain DNA fragments of 100–

400 nt. Chromatin immunoprecipitations were performed with an

antibody directed against RNAPII (catalog no. sc-899, Santa Cruz

Biotechnology). To test aspecific binding to the beads, a purified

IgG was used as a control for immunoprecipitation (catalog no. I-

1000, Vector Laboratories). Quantitative real-time PCR reactions

were performed using the MesaGreen qPCR mastermix (Euro-

gentec). Relative quantification using standard curve method was

performed for each primer pair and 96-well Optical Reaction

plates were read in an Applied Biosystems AbiPrism 7300 real-

time PCR instrument (Absolute Quantification Method). Fold

enrichments were calculated as percentages of input values

normalized to an unrelated genomic region (18S rRNA DNA)

and expressed as fold inductions relative to the values measured in

mock-treated U1 cells. Primer sequences used for quantification in

a region overlapping the LTR U5 region and the primer binding

site region (to avoid interference with the 3’LTR region) (FW, 59-

TGGAAAATCTCTAGCAGTGGC-39 and RV, 59-GAGTCC-

TGCGTCGAGAGATCT-39) and in the unrelated 18S rRNA

DNA region (FW, 59-TGGATACCGCAGCTAGGAATAA-39

and RV, 59-CCTCTTAATCATGGCCTCAGTTC-39) were

designed using the software Primer Express 2.0 (Applied

Biosystems).

RNA extractions and analyses of initiated and elongatedHIV-1 transcripts

Total RNA samples were isolated using the RNeasy Plus kit

(Qiagen) from 16106 U1 cells and digested with TURBO DNase

(TURBO DNA-freeTM kit, Ambion). First strand cDNA was

synthesized using SuperScript III Reverse Transcriptase (Invitro-

gen). Quantitative real-time PCR reactions were performed using

the MesaGreen qPCR mastermix (Eurogentec). Initiated tran-

scripts were detected with primers tar (FW, 59-GTTAGACCA-

GATCTGAGCCT-39 and RV, 59-GTGGGTTCCCTAGT-

TAGCCA-39). Elongated transcripts were detected with three

different sets of primers: pol (FW, 59-AATATGCAAGAAT-

GAAGGGTGC-39 and RV, 59-TGCTTTCTGTGGCTATTT-

TTTGTA-39), vif (FW, 59-ATGGCAGGTGATGATTGTGTG-

39 and RV, 59-CCATGTGTTAATCCTCATCCTGTC-39) and

nef (FW, 59-GCCTGGCTAGAAGCACAAGA-39 and RV, 59-

AGGTGTGACTGGAAAACCCAC-39). b-actin mRNA copies

were quantified with primers b-actin (FW, 59-GTCGA-

CAACGGCTCCGGC-39 and RV, 59-GGTGTGGTGCCA-

GATTTTCT-39) specific for a 239 bp region in the b-actin

mRNA. Quantitative real-time PCR reactions were performed for

each primer pair and 96-well Optical Reaction plates were read in

an Applied Biosystems StepOnePlus Real-Time PCR System

(Comparative Ct (DDCt) Quantification Method).

Flow cytometry analysesJ-Lat 8.4 or 15.4 cells were mock-treated or treated for 24 h

with the different compounds alone or in combination. Reco-

verded cells were washed twice in PBS, resuspended in PBS

containing 4% paraformaldehyde and fixed for 1 h. Cells were

next washed twice in PBS and resuspended in FACS buffer (PBS-

BSA 0.1%-NaN3 0.1%). The percentage of GFP-positive cells was

measured on a CXP cytometer (Cytomics FC 500, Beckman

Coulter) using CXP Software version 1.0 according to the

manufacturer’s instructions.

Supporting Information

Figure S1 Dose-response curves of U1 cellular viability after

HDACI treatment. U1 cells were mock-treated or treated with

increasing concentrations of an HDACI belonging to one of the

four structural families: short-chain fatty acids (A), benzamides (B),

cyclic tetrapeptides (C), hydroxamic acids (D). At 24 h posttreat-

ment, cellular viability was tested by measuring the mitochondrial

dehydrogenase activity with the WST-1 reduction assay. The

mock-treated value was arbitrarily set at a value of 100% of

cellular viability. Each point is the mean from three separate

experiments performed in triplicate. SE are intentionally not

represented on the graph for clarity reasons.

Found at: doi:10.1371/journal.pone.0006093.s001 (0.19 MB TIF)

Figure S2 Prostratin does not increase HDACI cytotoxicity in

CD8+-depleted PBMC cultures from uninfected individuals.

CD8+-depleted PBMCs were mock-treated or treated with VPA

(2.5 mM), SAHA (2.5 mM), TSA (500 nM), NaBut (5 mM), MS-

275 (5 mM), prostratin (5 mM) alone (A) or in combination (B). At

24 h posttreatment, cellular viability was tested by measuring the

mitochondrial dehydrogenase activity with the WST-1 reduction

assay. A value of 100% of cellular viability was arbitrarily assigned

to the mock-treated value (A) or to the prostratin-treated value (B).

Each value is the mean +/2 SE from three separate experiments

performed in triplicate.


Figure S3 Prostratin+HDACI cotreatment induces HIV-1

expression in a higher proportion of cells than the drugs alone.

This figure shows as plots the same FACS results that are

presented as histograms in Figure 3B in the manuscript. J-Lat 8.4

cells were mock-treated or treated with prostratin (5 mM), alone or

in combination with different HDACIs [VPA (2.5 mM), SAHA

(2.5 mM), TSA (500 nM), NaBut (5 mM) or MS-275 (5 mM)]. At

24 h posttreatment, cells were analyzed by FACS for GFP

expression. The plots are representative of four independent

experiments obtained with J-Lat 8.4 cells. Similar results were

obtained with the J-Lat 15.4 T-cell clone (data not shown).


Figure S4 The prostratin+VPA cotreatment causes a more rapid

and pronounced nucleosomal remodeling than the compounds

alone. (A) Diagram indicating the positions of nucleosomes in the

59 portion of the HIV-1 genome, the AflII and HinfI cutting sites

and the probe used in indirect end-labeling. Bold, lower case

letters are assigned to each HinfI cutting site (x, y and z) and are

located next to the bands on the gel to permit their identification.

(B) Nuclei were prepared from U1 cells mock-treated or treated

with TNFa (10 ng/ml) (30 min), prostratin (5 mM), VPA (2.5 mM)

and prostratin+VPA for 30 min, 1 h or 2 h and digested with

HinfI. After DNA purification and in vitro restriction with PstI,

DNA samples were analyzed by indirect end-labeling using probe

A (93). Size markers (a, b, c, d, e, f, g) have been previously

described (93).




Figure S5 The prostratin+VPA cotreatment does not induce

levels of acetylated histone H4 higher than the levels observed

after the treatments with VPA alone. Acetylated H4 levels in the

nuc-1 region were assessed by ChIP experiments using U1 cells

mock-treated or treated with prostratin (5 mM), VPA (2.5 mM)

and prostratin+VPA for different periods of time. The proteins

were cross-linked with formaldehyde for 10 min and DNA

sheared. The cross-linked protein/DNA complexes were immu-

noprecipitated with an anti-Ac-H4 antibody. The protein/DNA

cross-links were reversed and the purified DNA was amplified and

quantified by real-time PCR using primers amplifying either the

nuc-1 region or the vif region. Fold enrichments in the nuc-1 and

vif regions were calculated as percentages of input values and

expressed as fold inductions relative to the value measured with

the nuc-1 primers in mock-treated U1 cells, which was arbitrarily

set at a value of 1. Each value is the mean +/2 SE from three

separate experiments performed in duplicate.


Text S1 Supporting Information of Figure S1

Found at: doi:10.1371/journal.pone.0006093.s006 (0.08 MB

DOC)



DOC)



DOC)

Acknowledgments

We would like to thank the HIV-1-positive patients for their willingness to

participate in this study and Elodie Goudeseune who cares for the patients.

We thank Christelle Cardona, Noemie Stache and Adeline Melard for

excellent technical assistance. The following reagents were obtained

through the AIDS Research and Reference Reagent Program, Division

of AIDS, NIAID, NIH: the U1 cell line from Dr. T. Folks, the J-Lat Full

Length Clones (clones #8.4 and #15.4) from Dr. E. Verdin and the Jurkat

Clone E6-1 from Dr. A. Weiss.

Author Contributions

Conceived and designed the experiments: CVL. Performed the experi-

ments: SR MC AG JSG VQ CV AL DD VM EV TC VA SP JP YdL AB

CR OR YEC OL. Analyzed the data: SR MC OL CVL. Contributed

reagents/materials/analysis tools: YEC. Wrote the paper: SR MC CVL.

Performed measurements of HIV-1 genomic RNA concentrations: DV

GH. Performed patient selection: KK MM SDW OL NC. Performed the

isolation of HLA DR- CD4+ T lymphocytes: AL OL.

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